The present invention relates to vanadium complexes of monohydroxamates and to pharmaceutical compositions comprising them useful for the treatment of diabetes.
ABBREVIATIONS Asp(xcex2)HXM, L-aspartic acid xcex2-monohydroxamate; CytPTK, cytosolic protein-tyrosine kinase; Glu(xcex3)HXM, L-glutamic acid xcex3-monohydroxamate; HXM, monohydroxamate; InsRTK, insulin receptor tyrosine kinase; IRS-1, Insulin receptor substrate 1; PTK, protein tyrosine kinase; KRB, Krebs Ringer bicarbonate; NaVO3, sodium metavanadate; STZ, streptozocin; VOSO4, vanadyl sulphate; VOCl2, vanadyl chloride.
Intensive studies have been carried out in the last two decades on the insulinomimetic effects of vanadium (reviewed in Shechter et al.,1995). In vitro, vanadium salts mimic most of the effects of insulin on the main target tissues of the hormone, and in vivo they induce normoglycemia, and improve glucose homeostasis in insulin-deficient and insulin-resistant diabetic rodents (reviewed in Brichard and Henquin, 1995). In the basic research frontier, data continues to accumulate showing that vanadium salts manifest their insulin-like metabolic effects through alternative pathways, not involving insulin-receptor tyrosine kinase activation, nor phosphorylation of insulin-receptor substrate 1 (IRS-1). The key players of his back-up system appear to involve inhibition of protein-phosphotyrosine phosphatases and activation of nonreceptor protein tyrosine kinases (reviewed in Brichard and Henquin, 1995).
Vanadium is an ultra trace element in mammals. The dietary intake varies between 10-60 xcexcg dayxe2x88x921, and the intracellular concentration is approximately 20 nM (reviewed in Shechter et al., 1995 and Brichard and Henquin, 1995). The bulk of the intracellular vanadium is probably in the vanadyl (+4) form. Acute oral administration of vanadium compounds proved to be moderately toxic. Nevertheless, the profound anti-diabetic effects of oral vanadium therapy in insulin-deficient and insulin-resistant diabetic rodents (reviewed in Brichard and Henquin, 1995) encouraged the start of clinical studies. Small doses of vanadium (100-125 mg/person/day, over a period of 3 weeks) were allowed and, although 100 fold lower than those used in most animal studies, several beneficial effects were observed (Cohen et al., 1995).
Organically chelated vanadium (+4) complexes are about 5 fold more potent than free vanadium (+4) in facilitating the metabolic effects of insulin in vitro (Li et al., 1996) and in STZ-rats in vivo (Sakurai et al., 1995). The theoretical basis for that is still under study, and is likely the result of stabilizing the more potent insulin-like vanadium species.
Vanadium salts mimic the metabolic effects of insulin through alternative insulin-independent machinery components in vitro and can overcome states of insulin-resistance in diabetic rodents in vivo . As such vanadium therapy could make a useful, attractive and complementary approach to insulin therapy, provided that vanadium salts were less toxic or certain manipulation could be made to carry on vanadium therapy with no, or reduced quantities of, exogenous vanadium source.
Israel Patent No. 99666 and corresponding U.S. Pat. No. 5,338,759 of the same applicants describe vanadyl complexes of dihydroxamates of the formula:
R2R3C{CH2O(CH2)mCO[NHCHR(CH2)qCO]nNOHR1}2
which are said to be useful for the treatment of diabetes, but were later found by the inventors to be unsuitable in vivo for normalizing blood glucose levels in model diabetic rats.
Hydroxamic acid derivatives were shown to be involved in the microbial transport of iron, and therefore suggested for treatment of conditions of iron deficiency. They are also inhibitors of urease activity and indicated for the treatment of hepatic coma. Much of their biological activities are related to their potency to chelate a variety of metals. In the majority of metal chelates formed by hydroxamic acids, coordination occurs by deprotonation of the OH group and subsequent (O,O) coordination with the carbonyl oxygen and deprotonated OH.
Amino acid monohydroxamates are simple, nontoxic derivatives of amino acids. D-Aspartic acid xcex2-hydroxamate (D-Asp(xcex2)HXM) was shown to have antitumoral activity on murine leukemia L5178Y, both in vitro and in vivo, and is active against Friend leukemia cells in vitro (Tournaire et al., 1994). L-Glutamic acid (xcex3)-monohydroxamate (Glu(xcex3)HXM) is cytotoxic against leukemia L1210 cells in vitro, and against leukemia L1210 and melanoma B16 cells in vivo (Vila et al., 1990).
It has now been found in accordance with the present invention that certain amino acid monohydroxamates (HXM), in particular the L-forms of glutamic acid xcex3-monohydroxamate (Glu(xcex3)HXM) and aspartic acid xcex2-monohydroxamate (Asp(xcex2)HXM), interact with vanadium (+4) and vanadium (+5). At a 1:1 or 2:1 HXM: vanadium molar stoichiometry, they largely potentiate the insulinominetic potencies of vanadium (+4) and (+5) in vitro, and normalize the blood glucose level of streptozocin-treated rats in vivo.
The present invention relates to novel vanadium complexes of monohydroxamates of the formula (I):
Rxe2x80x94COxe2x80x94NHOH.Xxe2x80x83xe2x80x83(I)
wherein
R is a residue selected from:
(i) H2Nxe2x80x94CH (COY)xe2x80x94(CH2)nxe2x80x94
(ii) H2Nxe2x80x94CH (COOH)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94; and
(iii) pyridyl, piperidyl or tetrahydroisoquinolinyl;
wherein n is 1, 2 or 3, and Y is OH or NH2; and X is a vanadium compound selected from a vanadyl (VO2+), metavanadate (VO3xe2x88x92) or vanadate (VO43xe2x88x92) salt.
According to the present invention, in the monohydroxamates of (i) above, n is preferably 1 or 2, namely the xcex2- and xcex3-monohydroxamates of L-aspartic acid and L-glutanic acid, respectively. In a most preferred embodiment, the amino acid monohydroxamate is Glu(xcex3)HXM, that was found to be more effective in the manifestation of the metabolic effects of insulin in rat adipocytes as compared to various xcex1-amino acid monohydroxamates.
Interestingly enough, among all known vanadium chelators disclosed in the literature such as acetylacetonate, bispicolinato and the dihydroxamate RL-252 described in the above-mentioned U.S. Pat. No. 5,338,759, amino acid monohydroxamates are unique in their capacity to produce insulin effects, in vitro, in the absence of externally added vanadium, indicating that amino acid monohydroxamates might be capable of converting the minute quantity of intracellularly located vanadium (+4, xcx9c20 nM) into an insulinomimetically-active species. Further studies in vitro revealed that Glu(xcex3)HXM facilitates all the physiologically relevant bioeffects of insulin. These include activation of hexose uptake and inhibition of isoproterenol mediated lipolysis. Quite importantly, activation by Glu(xcex3)HXM is additive to maximal effect produced by a saturated concentration of insulin.
Among the monohydroxamates of (iii) above, preferred are the 3-pyridyl radical, namely the nicotinic acid hydroxamate, the 2- or 3-piperidyl radical and the 3-tetrahydroisoquinolinyl radical.
The monohydroxamates used in the present invention are soluble in water in contrast to the dihydroxamates of the above mentioned U.S. Pat. No. 5,338,759, that are water insoluble. Thus the vanadium complexes of the present invention can be prepared by simple dissolution in water of the monohydroxamate and of the vanadium salt.
Examples of vanadium salts used to form the complexes used in the compositions of the present invention are, without being limited to, VOCl2 (+4), VOSO4 (+4), NaVO3 (+5) and Na3VO4 (+5).
Various HXM: vanadium salt stoichiometric molar ratios of the complexes are envisaged by the present invention, but 1:1 and 2 HXM: 1 vanadium salt molar ratios are preferred.
The complexes of formula I of the invention are prepared by mixing water solutions of the monohydroxamate and the vanadium salt, freezing and lyophilizing the solution, thus obtaining a dry powder that can be stored, for example, at room temperature.
The invention further provides a pharmaceutical composition useful for the treatment of diabetes, particularly for reduction of blood glucose levels and induction of normoglycemia in diabetic patients, comprising a vanadium complex of a monohydroxamate of formula (I) as an active ingredient and, optionally, a pharmaceutically acceptable carrier. Said compositions may be used for treatment of both insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus NIDDM).
The dosage to be administered will depend on the conditions of the diabetic patient and may be within a range of from 0.2 mg/kg to 2 mg/kg daily. Since the allowed amount of vanadium being used in diabetic patients in clinical trials today is about 2 mg/kg/day, the minimum amount provided by the present invention represents a 10 fold increase in efficacy.
The compositions of the invention comprising the vanadium complex of formula I may be presented in soluble form, such as drops, or in the form of capsules or tablets and are preferably administered orally. They may be administered alone or in combination with insulin.
The vanadium complex of formula I may also be generated in vivo by separate administration of the vanadium salt and of the monohydroxamate. The invention thus further comprises a pharmaceutical package comprising a pharmaceutical composition comprising a monohydroxamate of the formula Rxe2x80x94COxe2x80x94NHOH, wherein R is a residue selected from: (i) H2Nxe2x80x94CH (COY)xe2x80x94(CH2)nxe2x80x94; (ii) H2Nxe2x80x94CH (COOH)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94; and (iii) pyridyl, piperidy or tetrahydroisoquinolinyl; and n is 1, 2 or 3, and Y is OH or NH2; and a pharmaceutical composition comprising a vanadium compound selected from a vanadyl (VO2+), metavanadate (VO3xe2x88x92) or vanadate (VO43xe2x88x92) salt, with instructions how to administer them. Preferably, the composition comprising the vanadium salt is administered prior to the monohydroxamate composition. The two ingredients may also be comprised within a compartment of a single composition, e.g. a capsule, separated by a non-permeable membrane.
Since a monohydroxamate of formula Rxe2x80x94COxe2x80x94NHOH above, particularly Glu(xcex3)HXM, can by itself associate to endogenous intracellular vanadium, modifying the latter into an active species that evoke the metabolic responses of insulin, the invention further envisages a pharmaceutical composition for the treatment of diabetes comprising a monohydroxamate of the formula Rxe2x80x94COxe2x80x94NHOH, wherein R is a residue selected from: (i) H2Nxe2x80x94CH (COY)xe2x80x94(CH2)nxe2x80x94; (ii) H2Nxe2x80x94CH (COOH)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94; and (iii) pyridyl, piperidyl or tetrahydro-isoquinolinyl; and n is 1, 2 or 3, and Y is OH or NH2.
The invention further relates to the use of a monohydroxamate of the formula Rxe2x80x94COxe2x80x94NHOH, wherein R is a residue selected from: (i) H2Nxe2x80x94CH (COY)xe2x80x94(CH2)nxe2x80x94; (ii) H2Nxe2x80x94CH (COOH)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94; and (iii) pyridyl, piperidyl or tetrahydro-isoquinolinyl; and n is 1, 2 or 3, and Y is OH or NH2, for the preparation of a pharmaceutical composition for the treatment of diabetes.
In another embodiment, the invention relates to a method for reduction of blood glucose levels in a diabetic patient which comprises administering to said patient an effective amount of a vanadium complex of a monohydroxamate of formula (I) herein, or of effective amounts of a monohydroxamate of the formula Rxe2x80x94COxe2x80x94NHOH herein and a vanadium compound selected from a vanadyl (VO2+), metavanadate (VO3xe2x88x92) or vanadate (VO43xe2x88x92) salt, or of effective amounts of a monohydroxamate of the formula Rxe2x80x94COxe2x80x94NHOH herein, alone or in combination with insulin therapy.
In a further embodiment, the invention relates to a method for inducing normoglycemia in a diabetic patient which comprises administering to said patient an effective amount of a vanadium complex of a monohydroxamate of formula (I) herein, or of effective amounts of a monohydroxamate of the formula Rxe2x80x94COxe2x80x94NHOH herein and a vanadium compound selected from a vanadyl (VO2+), metavanadate (VO3xe2x88x92) or vanadate (VO43xe2x88x92) salt, or of effective amounts of a monohydroxamate of the formula Rxe2x80x94COxe2x80x94NHOH herein, alone or in combination with insulin therapy.